1. Field of the Invention
The present invention relates to cell surface proteins on bone marrow stromal stem cells (BMSSCs), in particular CD18, which can be used as a marker for making stem cell enriched extracts from unfractionated bone marrow aspirates and from other sources such as adipose tissue. The invention also relates to methods of using the BMSSCs for the treatment of diseases, especially bone marrow diseases.
2. Description of the Related Art
Bone marrow contains stem-like precursors for non-hematopoietic cells, such as osteoblasts, chondrocytes, adipocytes and myoblasts (Owen et al., 1988, in Cell and Molecular Biology of Vertebrate Hard Tissues, Ciba Foundation Symposium 136, Chichester, UK, pp. 42-60; Caplan, 1991, J. Orthop. Res 9:641-650; Prockop, 1997, Science 276:71-74). Non-hematopoietic precursors of the bone marrow have been variously referred to as colony-forming-unit-fibroblasts, mesenchymal stem cells, and bone marrow stromal stem cells (BMSSCs). BMSSCs are believed to participate in the creation of the microenvironment within the bone marrow in vivo. When isolated, BMSSCs are initially quiescent but eventually begin dividing so that they can be cultured in vitro. Expanded numbers of stromal cells can be established and maintained. Stromal cells have been used to generate colonies of fibroblastic adipocytic and osteogenic cells when cultured under appropriate conditions. They can also be made to differentiate into cartilage cells and myoblasts.
There are several examples of the use of BMSSCs for treatment of disease. Stromal cells have been used to produce fibrous tissue, bone or cartilage when implanted into selective tissues in vivo (Ohgushi et al., 1989, Acte. Orthop. Scand. 60:334-339; Nakahara et al., 1992, J. Orthop. Res 9:465-476; Niedzwiedski et al., 1993, Biomaterials 14:115-121; and Wakitani et al., 1994, J. Bone & Surg. 76A:579-592). In some reports, stromal cells have been used to generate bone or cartilage in vivo when implanted subcutaneously with a porous ceramic (Ohgushi, et al., 1989, Acta Orthop. Scand. 60:334-339), intraperitoneally in a diffusion chamber (Nakahara et al., 1991, J. Orthop. Res 9:465-476), percutaneously into a surgically induced bone defect (Niedzwiedski, et al., 1993, Biomaterials 14: 115-121), or transplanted within a collagen gel to repair a surgical defect in a joint cartilage (Wakitani et al., 1994, J. Bone Org. 76A: 579-592). Piersma et al. (1983, Brit. J. Hematol. 94:285-290) disclose that after intravenous bone marrow transplantation, the fibroblast colony-forming cells which make up the hemopoietic stroma lodge and remain in the host bone marrow. Stewart et al. (1993, Blood 81:2566-2571) recently observed that unusually large and repeated administrations of whole marrow cells produced long-term engraftment of hematopoietic precursors into mice that had not undergone marrow ablation. Also, Biennzle et al. (1994, Proc. Natl. Acad. Sci. USA, 91:350-354) successfully used long-term bone marrow cultures as donor cells to permanently populate hematopoietic cells in dogs without marrow ablation. In some reports, stromal cells were used either as cells that established a microenvironment for the culture of hematopoietic precursors (Anklesaria, 1987, PNAS USA 84:7681-7685) or as a source of an enriched population of hematopoietic stem cells (Kiefer, 1991, Blood 78(10):2577-2582).
This new research has shown that adult stem cells in fact possess much wider potential for differentiation than previously thought. The identification and isolation of the stem cells enables the reinfusion of these long-term repopulating stem cells in various clinical therapies. For example, purging bone marrow stem cells of contaminating tumor cells would require reintroduction of new uncontaminated stem cells. In gene therapy, stem cells either from a patient or a donor can be transfected to contain new genes of therapeutic use and then reintroduced into the patient. Identification of surface markers on stem cells is extremely useful in hematopoietic research and related therapies because such markers allow the isolation of relatively pure populations of immature stem cells. However, the lack of appropriate surface markers has been a major obstacle for incorporating bone marrow stromal stem cells (BMSSCs) in clinical applications.(4) At present the only useful marker for this purpose is STRO-1. Thus, there is a continued need for the identification other antigens on BMSCCs to simplify the identification and separation of these cells from bone marrow aspirates (or from peripheral blood or adipose tissue which also have BMSSCs).